1. Field of the Invention
The present invention generally relates to a method and apparatus for providing fluorescence lifetime images and more particularly to a method and apparatus for providing multi-dimensional fluorescence lifetime imaging, using the phase-shift and/or modulation of the fluorescence signal. Also described is a method to suppress the fluorescence signal due to background and/or autofluorescence from the sample, or to visualize regions of the sample with decay times greater or less than a desired value.
2. Description of the Background Art
Quantitative fluorescence image analysis has application to a wide variety of practical arts including cellular physiology and biological and clinical research, immunology, chromosome analysis, environmental science, forensic analysis, fingerprint imaging and the like. However, significant problems have been encountered in such applications.
The availability of a variety of quantitative techniques for the evaluation of the chemical composition of specimens is of significant importance to the conduct of basic biological research and to a wide variety of clinical applications. Research involving the study and analysis of cells, generally known as cytology, employs a variety of analytical techniques for identifying and enumerating the subpopulations of cells in a specimen under study. For example, cytological materials may be examined to detect the presence of cancerous or malignant cells, or to determine the chemical composition of cells within a specimen. For purposes of analysis, the cells may be labeled with a variety of fluorescent materials, commonly known as probes or fluorophores, which have an identified affinity for cells or cell components which are of interest to an analysis. The probes will emit a particular fluorescence when stimulated by light at a known wavelength. The emitted light may have distinguishing characteristics, particularly wavelength and intensity, which permit an analysis of a subpopulation of cells or region of a cell to be conducted. The wavelength and intensity are dependent on the concentration of the analyte or cell component but, due to background or autofluorescence, measurements based on intensity or wavelength are limited to detection of analytes or compounds that are present at the highest concentrations in a cell or sample.
The study of collections of multiple cells using fluorescence spectroscopy has obvious additional problems such that an accurate determination of the number of cells in a subpopulation which have a given characteristic usually cannot be made since it is difficult to separate the subpopulations for analysis.
In one conventional approach using fluorescence microscopy (FM), specimens are tagged with fluorescent agents which bind or react with particular components of a specimen or cell component, and which are responsive to light emitted by a non-modulated light source at a characteristic wavelength to which the agent is sensitive.
When the fluorescent agent, bound to or reacted with a specimen, is excited by light at the agent's absorption wavelength, the energy level of an electron of the agent is raised above the relaxed or ground state to an excited state. Following excitation, the agent's electrons return to their relaxed state and emit light having a characteristic wavelength. Multiple agents may be used, each sensitive to light at the same or different wavelengths and each responsive to the stimulating light by emitting light at a characteristic wavelength that can be detected and used for analysis. Each agent can be responsive to various chemicals or biological molecules within the cell.
In a conventional FM system, as a particular fluorophore emits light following its excitation, a two-dimensional intensity image may be produced that is proportional to the local concentration of the fluorescent species having the characteristic wavelength which is detected. The areas having the highest intensity light emissions are detectable and identifiable as areas having the highest concentration of the related probe. The areas of less concentration of analyte, however, are not detectable, due to, e.g., high background fluorescence or autofluorescence. However, the concentration of the probe is often not of interest, but, rather, the important parameters include the concentration of an analyte to which the probe is responsive.
Thus, one major problem of such conventional two-dimensional fluorescence intensity imaging methods is that the measurement is concentration-dependent and often is not of interest or may be inaccurate due to the difficulty with fluorophore bleaching (i.e., a degradation of the fluorophore caused by the intensity of the incident laser light). The limitation of concentration-dependent measurements and the problem of photobleaching preclude accurate characterization of the chemical and physical properties of a specimen by conventional FM techniques. Additional limitations include the above-mentioned background and autofluorescence, as described in greater detail below.
The problem of photobleaching and the effects of probe concentration-dependent intensities are sometimes circumvented using probes which display wavelength shifts in response to the chemical species of interest. These probes were developed because conventional FM measurements are performed only as stationary measurements, and wavelength shifts are needed to cancel concentration and photobleaching effects. Additionally, few such wavelength-ratio probes are available, and these require excitation with ultraviolet light which causes problems including increased autofluorescence, cost and complexity, due to the general unavailability of ultraviolet lasers. These significant technical problems arise due to the need for a probe to display a special shift in order to eliminate concentration effects.
As seen in U.S. Pat. No. 4,778,539, Yamashita et al, issued Oct. 18, 1988, individual cells may be distinguished by measuring light at an observation point of a flow cytometer system. There, the change in the intensity of transient emitted light over a period of time, following excitation by short pulses of laser light, may be measured and used to detect the attenuation time of the emitted light, the rise time of the emitted light and the orientation relaxation time. These parameters may be used as a basis for cell discrimination. However, such measurements are performed cell-by-cell, and do not allow for rapid and/or simultaneous scanning of a population of cells, and do not allow for distinguishing regions within a cell.
There have been some attempts to measure lifetimes using a microscope and laser to provide site-selective measurements. See, for example, Fernandez Biophys. J., 37:73a (1982); Ramponi and Rodgers Photochem. and Photobiol., 45:161-165 (1987); Docchio et al J. Microscopy, 134:151-160 (1983); and Keating and Wensel SPIE, 1204:42-48 (1990). However, such measurements are complex even when performed at a single site, and these methods are not easily extended to two dimensional imaging. For instance, the technique of photon-counting lifetime measurements by timeresolved fluorescence through a microscope has been extended by scanning single pixels by Wang et al (Applied Spectroscopy, 44:25-30 (1990)) to provide two-dimensional mapping of samples. However, these images are limited to the use of spot-by-spot, or pixel-by-pixel, measurements under the microscope.
Phase-modulation fluorescence spectroscopy (PMFS) provides further means by which fluorescence lifetime selectivity can be implemented in a static environment. See J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press (1983), which teaches a technique in which a sample is excited with light having a periodic time-dependent intensity, and detection is made of the resulting time-dependent emission. Because the emission is demodulated and phase shifted to an extent determined by the fluorescence lifetime of the species, the fluorescence lifetime (.tau.) can be calculated from the phase shift of the species: ##EQU1## or from a demodulation factor ##EQU2## where m is a demodulation factor, .omega. is the angular modulation frequency and .phi. is the phase shift of the species. A modification of PMFS is the technique of phase-sensitive detection or phase-resolved fluorescence spectroscopy (PRFS), which results from a comparison of the detected emission with an internal electronic reference signal of the same frequency. phase-sensitive detection can be accomplished by using the high light- modulation frequencies (see, e.g., Veselova et al Opt. Spect., 29:617-618 (1970) and Veselova and Shirokov Akad. Nauk. SSSR Bull. Phys. Sci. 36:925-928 (1972)) and more easily at lower cross-correlation frequencies. See Lakowicz and Cherek J. Biochemo Biophys. Methods, 5:19-35 (1981).
In phase-resolved fluorometry, the time-dependent fluorescence photocurrent is multiplied by a periodic square-wave signal which has the same modulation frequency as the fluorescence signal, in order to fully detect an output signal, and is then integrated. A time-independent DC signal is thereby produced that is proportional to the cosine of the difference between the phase angles of the square wave and the fluorescence signal, and proportional to both signal amplitudes. However, it is important to recognize that the output signal in phase-resolved fluorescence is still proportional to the signal intensity.
The use of phase-resolved methods has been suggested for obtaining a scanning measurement of single pixels to generate a two-dimensional fluorescent measurement. For instance, Wang et al (Applied Spectroscopy, 43:840-845 (1989)) suggested a fluorescence lifetime distribution measurement system using an image dissector tube with phase-resolved detection. However, such methods suffer from the problem that measurements can be obtained only at a single spot or pixel, and the image is constructed by scanning of the spot.
A further problem of such scanning fluorescence measurements is that background and/or autofluorescence can each contribute to poor image contrast, particularly where the intensity level of fluorescent signals is low or the contrast between different fluorescent signals is small.
Attempts have been made to practically detect and diagnose early stage cancer by hematoporphyrin derivative (HpD) fluorescence analysis. HpD is an effective photosynthesizer in photodynamic therapy for treatment of a variety of solid malignant tumors in man. Picosecond fluorescence spectroscopy, however, has shown that the major detectable HpD fluorescence originates from monomers that show no tumor localizing ability. In contrast, the HpD which accumulates in cancerous tissue exists primarily as aggregates which are only weakly fluorescent. Hence, even though HpD aggregates accumulate preferentially in cancerous tissue, the relative increase in intensity is modest, due to the influence of background and auto fluorescence. Thus, such a method of quantitative image analysis suffers from a low contrast of fluorescence from cancer cells as compared to that from normal cells.
In an attempt to overcome such problems, blue-violet alternating-wavelength fluorescence excitation may be used to increase the contrast. However, such a corresponding system is very complex and has limited practical applicability. The ability to obtain image contrast based on lifetimes rather than intensity may allow superior localization of cancerous tissues.
Background and auto-fluorescence also present a problem when latent fingerprints are detected by laser-excited fluorescence using techniques that are now employed by law enforcement agencies world-wide. Such techniques for the detection of fingerprints via the inherent fingerprint fluorescence suffer from the problem that suitable detection is possible only on surfaces that display little or no background fluorescence. However, most surfaces of practical interest show intensive autofluorescence which can make direct fingerprint detection impractical or impossible.
Fingerprint and other fluorescence imaging techniques can achieve autofluorescence signal suppression, or superior contrast between regions of interest, by utilizing time-resolved imaging. For example, because many autofluorescent surfaces of practical interest display fluorescence lifetimes as short as 0.1 ns to about 2 ns, fingerprints may be treated with staining dyes that have extremely long fluorescent lifetimes, typically 1 .mu.s or longer. By applying square wave-modulated laser excitation, the fast-decaying autofluorescence signal can be suppressed by means of an image intensifier with delayed ON-gating. However, the technique of pulsed image intensifier gating is restricted to fluorescence lifetimes that differ by at least ten-fold, in order to allow for an efficient lifetime-based selective signal suppression. For lifetimes that differ by less than ten-fold, e.g., by a factor of two, no significant contrast enhancement can be expected.
Moreover, only the signal corresponding to the component with the shorter lifetime can be suppressed entirely. The signals of the component having the longer lifetime can be suppressed only in part because they are time-overlapping with the fast-decaying signal.
In general, however, optional and complete suppression of either the fast-decaying or the slow-decaying signal is of interest to distinguish between lifetimes of similar values. For instance, in the case of a HpD-based method of cancer detection, the HpD aggregates accumulating in cancerous cells exhibit a lifetime of 0.1 ns, and monomers that show no tumor localizing ability have a longer decay time close to 4 ns. In this case, one would be interested in suppressing the signal of the 4-ns component. However, efficient suppression of the long decay time component based on pulse-gating is not readily realized, because commercially available image intensifiers allow only for a minimum gating time of about 5 ns. Due to the rise and fall time of the gating characteristic, no efficient time-selective signal suppression can be expected if both fluorophores have lifetimes shorter than 5 ns. Hence, it is desirable to be able to selectively observe the long or short decay time components in the emission.
A sinusoidally modulated image intensifier, combined with a linear photodiode array, has been utilized to acquire time-resolved fluorescence spectral data, as reported by Gratton et al (SPIE, 1204:21 (1990), but this apparatus was not used for position-sensitive measurements or to create lifetime images. Also, radio-frequency phase-sensitive imaging has been performed by using a position-sensitive high-speed photodetector, combined with an electronic correlator. See SPIE, 1204:798 (1990).
A conventional image intensifier pulse-gating technique for time-resolved imaging is illustrated schematically in FIG. 1. The fluorescent target 1 under test is illuminated by ultrashort periodic laser pulses 2. Two small areas 3, 4, one with a long decay time .tau..sub.2 and one with a short decay time .tau..sub.1, are shown on the target. By applying properly delayed electrical square wave pulses 5 to the image intensifier 6, imaging of the fast-decaying fluorescent area 4 onto the phosphor screen 7 can be suppressed, yielding only an image 3'.
As discussed above, no efficient decay time-selective suppression is possible if both decay times are comparable, or if both are too short. Also, only incomplete imaging suppression is attainable for the area 3 showing a longer decay time, due to time-overlapping with the emission emerging from the area 4 with the shorter decay time. The decay time, when compared to the square wave pulse 5 input to the image intensifier, is illustrated in FIG. 1 for each of the long decay time 8 and short decay time 9.